The present invention relates to methods for purification of antithrombin-III (AT-III) by precipitation of impurities. The invention also relates to pharmaceutical compositions obtainable by the said methods.
Antithrombin III (AT-III) is a plasma glycoprotein that inhibits serine proteases in the coagulation cascade and thus plays a major role in the regulation of blood clotting. A small decrease of the AT-III content in the blood is associated with increased risk of thromboembolism. AT-III concentrates are used in the prophylaxis and treatment of thromboembolic disorders in patients with acquired or hereditary antithrombin deficiency.
Generally, AT-III is isolated from human plasma and administered to the bloodstream of the patient. Consequently, virus inactivation of AT-III concentrates is desirable. Precipitation with polyethylene glycol (PEG) has been widely used in AT-III purification for concentrating the protein and for precipitating viruses (see e.g. Wickerhauser et al. (1979) Vox Sanguinis 36, 281). In addition to PEG, also barium sulfate, ethanol, trichloroacetic acid, dextran- and ammonium sulfate have been used as precipitating agents during purification of AT-III. Many of these precipitating agents would be harmful if present in the final AT-III formulation. Consequently, subsequent removal of these agents is necessary, and the recovery of AT-III is thereby reduced.
PEG precipitation alone is not sufficient to ensure complete removal of hepatitis virus. AT-III concentrates for therapeutical use are therefore normally pasteurized, normally at +60xc2x0 C. for 10 h. In general, plasma proteins lose activity during heat treatment. For this reason, stabilizing agents are used during pasteurization.
Citrate and carbohydrates, such as sucrose, have been used as stabilizing agents for AT-III during pasteurization (Mitra et al. (1982) Biotechnology and Bioengineering vol. XXIV, 97-107; Tengborn et al. (1987) Thrombosis Research 48, 701-711; Einarsson et al. (1989) Transfusion 29, 148-152). However, there is no indication in these documents that citrate and saccharides can be used as precipitation agents in the purification of AT-III.
As a final step in the purification of AT-III, the formulation is often lyophilized. Many attempts have been made to prolong the shelf life of lyophilized AT-III.
U.S. Pat. No. 4,340,589 (Uemura et al.) discloses a lyophilized preparation of AT-III, stabilized with at least one substance selected from amino acids, saccharides, polysaccharides, etc., more specifically albumin, urokinase, gelatin, mannitol, heparin, glycine and lysine.
Ashizawa et al. (Japanese Patent Application No. 1994-199566) discloses lyophilized preparations of modified AT-III stabilized with one or more elements selected from organic acid salts, saccharides, amino acids and sodium chloride. The said organic salts could be sodium succinate or sodium citrate. The said saccharides could be D-mannitol, lactose, glucose and D-sorbitol. There is no indication in this document of lyophilized AT-III preparations comprising citrate in combination with sucrose.
A more or less known problem with liquid pharmaceutical preparations, is the formation of particles (approximately 2-75 um) after filling the product in vials or after reconstitution of freeze-dried products with a solution. Especially when working with larger molecules, like proteins, this is a phenomenon which relatively often occurs and the mechanism behind this is not clearly understood. Probably it is a combination of factors which affects in which amount and size the particles occurs, including molecular size of product, excepients of product, material of product container and solution in which the product is reconstituted in. It has not been shown that it should be of any danger having this relatively small amounts of particles in the products. However, it is obviously, that every pharmaceutical producer is aiming to reduce the amount of particles as much as possible in the products, especially the visible particles.
It has surprisingly been found that a combination of a saccharide, such as sucrose, and citrate can advantageously be used for precipitating impurities, including viruses and proteins other than AT-III, in the purification of AT-III. This procedure involves several advantages:
The precipitation step according to the invention assures high viral inactivation, high purity and minimal AT-III deactivation.
The obtained AT-III solution can be subjected directly to pasteurization without further addition of stabilizing compounds. Thus the combination of citrate and saccharide serves the dual purposes as precipitating agents and stabilizing agents during pasteurization.
There is no need to remove the pharmaceutically acceptable combination of saccharide and citrate during further purification of AT-III. Instead the precipitating agents are also useful as stabilizers of a lyophilized preparation of AT-III.
Consequently, in a first aspect this invention provides a process for purifying antithrombin-III comprising the steps of:
(a) adding, to a solution comprising antithrombin-III, a saccharide and citrate, in an amount sufficient for impurities in the said solution to precipitate while antithrombin-III essentially remains in solution;
(b) allowing impurities to precipitate; and
(c) removing the precipitated impurities, thereby obtaining a solution comprising purified antithrombin-III.
In the present context, the term xe2x80x9cimpuritiesxe2x80x9d is intended to mean undesired substances, including substances used during purification of AT-III (cf. Example 1, below). Impurities include e.g. histidine-rich glycoproteins, hemopexine, lipoproteins, gammaglobulins, Triton-X-100, tri-n-butyl-phosphate, virus, and prions.
The AT-III for use in the process according to the invention can be obtained by any suitable process and be of mammalian, e.g. bovine, porcine or, preferably, human origin. The AT-III may also be obtained by genetic engineering, such as by recombinant DNA techniques or from transgenic animals, e.g. from the milk of sheep producing AT-III in its milk.
Irrespective of origin, the AT-III can be of any isoform, e.g. xcex1-AT-III or xcex2-AT-III, or any derivative of AT-III. The difference between xcex1-AT-III or xcex2-AT-III is described e.g. by Brennan et al. (FEBS Letters 219(2), 431-436, 1987). The term xe2x80x9cderivative of AT-IIIxe2x80x9d refers e.g. to polypeptides carrying modifications like substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of AT-III.
The said saccharide is preferably a disaccharide such as sucrose or trehalose, or a monosaccharide such as glucose, sorbitol, mannitol, glyconic acid, or maltose. Sucrose is preferred because of its advantageous bioavailability in the body.
For precipitation of impurities, the concentration of saccharide can range from about 10% (w/v) to about 30% (w/v). Preferably the saccharide concentration is from between 15% and 25% (w/v), most preferably from about 20% (w/v).
The source of citrate can be citric acid or a pharmaceutically acceptable salt thereof, e.g. alkali metal citrates and alkaline earth metal citrates. Examples of alkali metal citrates are sodium and potassium citrate, while examples of alkaline earth metal citrates are magnesium and calcium citrate. For reasons of bioavailability, low cost and easy handling, sodium citrate is preferred.
For precipitation of impurities, the concentration of citrate can range from about 0.1 to about 3 M. Preferably the citrate concentration is from between 0.5 and 1.5 M, most preferably from between 1 M and 1.25 M.
During precipitation of impurities, pH can range from about 6 to about 9. Preferably, pH is from between 7 and 8, and most preferably neutral (around 7.5). The precipitation is suitably performed at a temperature from between +10xc2x0 C. and +40xc2x0 C., preferably at room temperature, such as from between +15xc2x0 C. and +25xc2x0 C.
Preferably, citrate and saccharide are added continuously to the solution under stirring during approximately 30 minutes. This procedure gives rise to a concentration gradient of saccharide and citrate. Since impurities might have a tendency to precipitate at different concentrations of the precipitating agents, such a gradient will optimize precipitation of impurities. A slow addition of the precipitating agents will also minimize the risk for high local concentrations of citrate and saccharide and thereby the risk for precipitation of AT-III.
In large-scale production, the solution is suitably stirred for approximately another 15 minutes after the final concentrations of saccharide and citrate have been reached.
The precipitated impurities are removed, suitably by filtration or centrifugation of the solution. A typical filtration step will comprise prefiltration trough a 1-20 xcexcm filter, followed by sterile filtration through a 0.2 xcexcm filter. If the choice is centrifugation, the skilled person will be able to determine suitable conditions with the aid of an ordinary textbook on protein purification. The filtrate or supernatant liquid comprising the antithrombin-III is then recovered in a form suitable for further processing.
In a further aspect, the invention comprises a process for purification of AT-III, wherein the precipitation step as described above is followed by pasteurization of the obtained solution comprising purified antithrombin-III, in the presence of a saccharide, in particular sucrose, and citrate as stabilizing agents. In a particularly preferred form of the invention, the already added amounts of saccharide and citrate will be sufficient as stabilizing agents during pasteurization, and consequently no further stabilizing agents will have to be added.
In yet a further aspect, the invention comprises a process for purification of AT-III as described above, in addition comprising lyophilization of antithrombin-III in the presence of a saccharide, in particular sucrose, and citrate as stabilizers. The same agents as previously used for precipitation and stabilization during pasteurization can conveniently be used also as stabilizers during lyophilization.
It will be understood by the skilled person that additional purification steps, subsequent to the pasteurization step, may be desirable to obtain AT-III in a form suitable for use or for lyophilization. Such steps can e.g. include, in any desired order:
Affinity chromatography, on a suitable medium such as Heparin-Sepharose(copyright), in order to remove inactivated AT-III;
Ultrafiltration in order to concentrate and desalt AT-III;
Filtration for further removal of virus.
The skilled person will be able to perform necessary further purification steps by methods well known in the art. An example of AT-III purification is given in Example 1, below.
In another aspect, this invention provides a pharmaceutical composition obtainable by the methods as described above. The composition according to the present invention may be used as a liquid preparation without further processing. However, prior to administration the liquid preparation is suitably further processed by drying AT-III in the presence of stabilizers. A suitable method for drying is lyophilization. Other possible methods for drying include e.g. vacuum concentration.
Consequently, in a preferred form of the invention, the composition is a lyophilized AT-III composition having increased stability (prolonged shelf-life) compared with known AT-III compositions. The said composition comprises (i) antithrombin-III; and (ii) sucrose and citrate as stabilizing agents. It will be understood by the skilled person that the composition could optionally comprise additional pharmaceutically acceptable excipients and/or carriers, such as salts, e.g. sodium chloride; amino acids, e.g. lysine, alanine, glycine or histidine; surfactants, e.g. polysorbate 80, also known under the trademark Tween-80(copyright); and other excipients such as polyethylene glycol (PEG 4000) or gluconic acid.
For preparation of a lyophilized AT-III composition according to the invention, a desired amount of the stabilizers is dissolved or preferably diafiltrated into an aqueous solution of AT-III having an appropriate concentration (from 50 to 1000 IU/ml, or from 5 to 200 mg/ml). The pH of the solution should be adjusted to from between 6 and 9, preferably from between 7 and 8. The solution is then sterile filtered, filled in vials, such as tubular glass bottles, and lyophilized.
The relative amount of sucrose in the composition is preferably from 0.5 to 2.5, more preferably from 1.0 to 1.5, parts per part antithrombin-III, by weight. The relative amount of citrate, preferably sodium citrate, is preferably from 1 to 4, more preferably from 2.5 to 3.0, parts per part antithrombin-III, by weight.
The AT-III composition according to the invention is further characterized in that it is stable without the addition of plasma-derived proteins, such as albumin. The absence of additional plasma-derived proteins increases the purity and viral safety in the composition of the invention.
When the pharmaceutical composition according to the invention is used to treat patients suffering from thromboembolic disorders, the lyophilized preparation can be dissolved in a physiologically isotonic salt solution or in sterile water, e.g. to a concentration of from 0.5 to 20% (w/v) of AT-III. The solution can be administered intravenously to obtain a systemic effect. It is, however, also possible to administer the solution to obtain a local effect, e.g. during balloon surgery of the coronary arteries.
As mentioned above, a more or less known problem with liquid pharmaceutical preparations, is the formation of visible particles (approximately 2-75 um) occurring after filling the product in vials, and particularly after reconstitution of freeze-dried products. Thus, a further aim of the present invention is to obtain a reconstituted products essentially free from such visible particles.
The inventor of the present application has surprisingly now found that adding a non-ionic surfactant to the reconstituted product in an amount of from at least 0.01% (w/w), provides a reconstituted AT-III product being essentially free from visible and undesired particles for a time period of as long as up to 3 months. The non-ionic surfactant is preferably selected from block co-polymers such as poloxamers, or polyoxyethylene sorbitan fatty acid esters, preferably polysorbate 80 (polyoxyethylene 20 sorbitan monooleate) which is also known under the trademark Tween 80(copyright), as the reconstitution media. Any type of polyoxyethylene sorbitan fatty acid ester may be used as reconstitution media in accordance with the present invention, as long as it is pharmaceutically and pharmacologically acceptable. Further examples of such polyoxyethylene sorbitan fatty acid esters suitable for use in accordance with the present invention are polysorbate 20 (polyoxyethylene 20 sorbitan monolaurate), also known under the trademark Tween 20(copyright); polysorbate 21 (polyoxyethylene(4)sorbitan monolaurate, also known under the trademark Tween 21(copyright); polysorbate 40 (polyoxyethylene 20 sorbitan monopalmitate), also known under the trademark Tween 40(copyright); polysorbate 60 (polyoxyethylene 20 sorbitan monostearate), also known under the trademark Tween 60(copyright); polysorbate 61 (polyoxyethylene (4) sorbitan monostearate), also known under the trademark Tween 61(copyright); polysorbate 65 (polyoxyethylene 20 sorbitan tristearate), also known under the trademark Tween 65(copyright); polysorbate 81 (polyoxyethylene (5) sorbitan monooleate), also known under the trademark Tween 81(copyright); polysorbate 85 (polyoxyethylene 20 sorbitan trioleate), also known under the trademark Tween 85(copyright); and polysorbate 120 (polyoxyethylene 20 sorbitan monoisostearate), also known under the trademark Crillet 6(copyright). Such polyoxyethylene sorbitan fatty acid esters are disclosed in Handbook of pharmaceutical excipients, 3rd edition, pp. 416-419, editored by Arthur H Kibble, which is hereby incorporated as reference.
Still another type of suitable surfactants useful in accordance with the present invention are sorbitan fatty acid esters. Any type of sorbitan fatty acid esters may be used as reconstitution media in accordance with the present invention, as long as it is pharmaceutically and pharmacologically acceptable. Suitable sorbitan fatty acid esters useful as reconstitution media in accordance with the present invention are disclosed in Handbook of pharmaceutical excipients, 3rd edition, pp. 511-514, editored by Arthur H Kibble, which is hereby incorporated in full as reference.
Experimental Methods
Biologic activity (IU/ml) of AT-III was determined as heparin cofactor activity by monitoring the cleavage of the chromogenic substrate H-D-Phe-Pip-Arg-pNA.2 HCl (Chromogenix, Sweden) by thrombin in presence of heparin and AT-III. See Frantzen Handeland et al. (Scand. J. Haematol. 31, 427-436, 1983) and van Voorhuizen et al. (Thromb. Haemostas. 52(3), 350-353, 1984).
Total protein concentration was determined by absorption measurements at 280 nm (A280) . Concentration (mg/ml) for AT-III solutions was calculated using the extinction coefficient of 6.4 IU/mg AT-III.
Specific activity of AT-III was defined as the ratio between heparin cofactor activity calculated as IU/ml and A280.
Dimer and polymer content of AT-III was determined by high performance liquid chromatography (HPLC) gel filtration on a TSK G 3000 SW column (Toyosoda) with 10 xcexcM particles and a pre-column (Toyosoda).
The amount of contaminating proteins was determined by crossed immuno-electrophoresis against anti-total human serum using standard conditions.
In order to determine that the reconstituted liquid pharmaceutical product is essentially free from visible particles, a sample is passing a detector, in which a laser source is transmitting light and a photosensor detects whenever a particle passes. The principle is called xe2x80x9clight block principlexe2x80x9d. The amplified impulse from the photosensor is collected by the particle counter (Hiac Royco) and thereafter converted to particle size and amount.